EPA Method 1694: Analysis of Pharmaceuticals and Personal Care Products in Water
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1 EPA Method 1694: Analysis of Pharmaceuticals and Personal Care Products in Water UCT Part Numbers ECHLD156-P ENVIRO-CLEAN HL DVB 500 mg, 6 ml cartridge VMF016GL 16 position glass block manifold VMFSTFR12 Large volume sample transfer tubes SLDA100ID21-3UM Selectra DA HPLC column ( mm, 3 µm) SLDAGDC21-3UM Selectra DA guard cartridge ( mm, 3 µm) SLGRDHLDR Guard cartridge holder Summary: EPA Method 1694 was published in December 2007 as a screening method for the analysis of pharmaceuticals and personal care products (PPCPs) in environmental samples, including water (wastewater, surface water and drinking water) [1]. The method uses solid-phase extraction (SPE) and liquid chromatography tandem mass spectrometry (LC-MS/MS) for the analysis of 73 PPCPs in water (a procedure is also outlined for the analysis of PPCPs in soil, sediment, and biosolids). The PPCPs include common prescription drugs, veterinary drugs, over the counter medicines, dietary supplements and other consumer products. The method divides the PPCPs into four groups based on their physicochemical properties. Water samples are extracted by means of two SPE procedures, using acidic (ph 2) and basic (ph 10) loading conditions. They are then analyzed by LC-MS/MS using four distinct methods according to their polarity and the extraction conditions of the PCPPs. LC-MS/MS analysis uses different LC conditions (column, mobile phase and gradient) and ionization modes (ESI + and ESI - ). EPA Method 1694 is not a regulatory method (i.e. 40 CFR Part 136) and instead is a guide that can be used to screen samples from various sources to profile the occurrence of PPCPs. It can also be used as a starting point for developing a custom PPCP method. Modifications to the method are allowed if they are documented and provide performance equal to or better than that specified in the method. This application note outlines a SPE procedure using UCT s Enviro-Clean HL DVB polymeric sorbent for the determination of 64 PPCPs in water according to EPA Method The SPE procedure was optimized with the aim of obtaining acceptable recoveries of the PCPPs using a single extraction step rather than the multiple extraction procedures outlined in the original EPA method. The LC-MS/MS analysis was also streamlined and uses only two methods (ESI + and ESI - ) and a single Selectra DA HPLC column instead of the four methods and two HPLC columns used in the original method. The unique chemistry of the Selectra DA column, which contains a polyaromatic stationary phase, provides a high degree of retention and selectivity for aromatic compounds and improved retention of polar compounds.
2 Sample Pretreatment: Because the analytes may be bound to suspended particles, the preparation of aqueous samples depends on the presence of visible particles. Aqueous samples absent visible particles can be analyzed directly according to the procedure outlined below. Aqueous samples with visible particles (>1%) should be filtered and the solids extracted and combined with the aqueous portion of the sample prior to the SPE procedure outlined below. Consult EPA Method 1694 for detailed information on sample collection, sample pretreatment, solids extraction, preparation and fortification of the target analytes and isotopically labeled internal standard solutions [1]. No ph adjustment of the sample is required. If residual chlorine is present, add 80 mg/l of sodium thiosulfate. Add 500 mg/l of tetrasodium EDTA (removes metal ions that can bind with the analytes). Spike sample with appropriate concentrations of internal standard and mix thoroughly (add target analytes for fortified samples). SPE Procedure: 1. SPE Conditioning a) 10 ml acetone. b) 10 ml reagent water, leaving approximately 2 ml of water on the top of the frit. 2. Sample Extraction a) Attach a large volume sample transfer tube (VMFSTFR12) to the top of each SPE cartridge and place the stainless steel end of the transfer tube directly into the sample bottle. b) Adjust the vacuum so that the flow rate is approximate 5-10 ml/min. 3. Wash Cartridge a) Remove the large volume transfer tubes and rinse the SPE cartridges with 10 ml reagent water (to remove EDTA). b) Dry the SPE cartridges under full vacuum (10-15 in. Hg) for 10 min. 4. Elution a) Elute the analytes with 10 ml methanol/acetone (1:1, v/v). Use a low vacuum such that the solvent exits the cartridge in a dropwise fashion. After the solvent has passed through, apply full vacuum for 30 seconds so that all the elution solvent is collected. 5. Concentration b) Evaporate extract to near dryness at C under a gentle stream of nitrogen. c) Reconstitute in 1 ml methanol. d) Vortex and transfer the sample to an autosampler vial for LC-MS/MS analysis.
3 LC-MS/MS Parameters: Instrumentation HPLC system Thermo Scientific TM Dionex TM Ultimate TM 3000 MS system HPLC column Guard column Guard column holder Column temperature 30 C Flow rate Injection volume Thermo Scientific TM TSQ Vantage TM (MS/MS) UCT Selectra DA, mm, 3 µm (p/n: SLDA100ID21-3UM) UCT Selectra DA, mm, 3 µm (p/n: SLDAGDC21-3UM) p/n: SLGRDHLDR 300 µl/min 5 µl (ESI + method) 10 µl (ESI - method) Time (min) Method 1 (ESI + ) Mobile Phase A (%) Water + 0.1% Formic Acid Mobile Phase B (%) Methanol + 0.1% Formic Acid Time (min) Method 2 (ESI - ) Mobile Phase A (%) Water + 10mM NH 4OAc Mobile Phase B (%) Acetonitrile
4 Retention Times and MRM Transitions Method 1 (ESI + ) Analyte RT Polarity Precursor Product 1 Product 2 Metformin Metformin-D Sulfanilamide Albuterol Albuterol-D Acetaminophen Acetaminophen-D Cotinine Cotinine-D Cimetidine Lincomycin Ranitidine Codeine Ampicillin Trimethoprim Trimethoprim- 13 C Sulfadiazine Sulfathiazole Oxytetracycline Ormetoprim Thiabendazole Thiabendazole-D Dimethylxanthine Minocycline Norfloxacin Tetracycline Sulfamerazine Ofloxacin Ciprofloxacin Ciprofloxacin- 15 N- 13 C Sulfamethizole Sulfamethoxazole Sulfamethoxazole- 13 C Cefotaxime Lomefloxacin Demeclocycline Azithromycin Sulfamethazine Sulfamethazine- 13 C Sulfachloropyridazine Enrofloxacin Chlortetracycline Sarafloxacin
5 Digoxigenin Doxycycline Diphenhydramine Sulfadimethoxine Fluoxetine Fluoxetine-D Caffeine Caffeine- 13 C Erythromycin Erythromycin- 13 C Carbadox Erythromycin anhydrate Anhydrotetracycline Penicillin G Atrazine- 13 C 3 (IIS) Clarithromycin Tylosin Diltiazem Penicillin V Carbamazepine Roxithromycin Oxacillin Digoxin Cloxacillin Oxolinic acid Miconazole Flumequine Virginiamycin Method 2 (ESI - ) Analyte RT Polarity Precursor Product 1 Product 2 TCPAA- 13 C 6 (IIS) Naproxen Naproxen-D Warfarin Warfarin-D Ibuprofen Ibuprofen- 13 C Gemfibrozil Gemfibrozil-D Triclocarban Triclocarban- 13 C Triclosan Triclosan- 13 C IIS = injection internal standard
6 Results and Discussion: Fortification EPA 1694 IPR* Criteria Deionized Water Results (n=6) Tap Water Results (n=6) Analyte Level (ng) Recovery (%) RSD (%) Recovery (%) RSD (%) Recovery (%) RSD (%) Acetaminophen Albuterol Ampicillin Anhydrotetracycline Azithromycin Caffeine Carbadox Carbamazepine Cefotaxime Chlortetracycline Cimetidine Ciprofloxacin Clarithromycin Cloxacillin Codeine Cotinine Demeclocycline Digoxigenin Digoxin Diltiazem Dimethylxanthine Diphenhydramine Doxycycline Enrofloxacin Erythromycin 15 N/A N/A Erythromycin anhydrate Flumequine Fluoxetine Gemfibrozil Ibuprofen Lincomycin Lomefloxacin Metformin Miconazole Minocycline Naproxen Norfloxacin Ofloxacin Ormetoprim Oxacillin Oxolinic acid Oxytetracyline Penicillin G Penicillin V Ranitidine Roxithromycin Sarafloxacin Sulfachloropyridazine Sulfadiazine Sulfadimethoxine Sulfamerazine Sulfamethazine Sulfamethizole Sulfamethoxazole Sulfanilamide Sulfathiazole Tetracycline Thiabendazole Triclocarban Triclosan Trimethoprim Tylosin Virginiamycin Warfarin *IPR = Initial precision and recovery
7 Relative Abundance RT: NL: 1.66E7 TIC MS Tap_water _ Time (min) Figure 1: TIC chromatogram of the ESI + analytes in an extracted tap water sample. RT: NL: 4.89E4 TIC MS Tap_H2O_ Time (min) Figure 2: TIC chromatogram of the ESI - analytes in an extracted tap water sample.
8 The original EPA 1694 method divides the PCPPs into four groups with each group having its own separate LC-MS/MS method. Group 1 contains the largest number of analytes; Group 2 is specific to the tetracyclines; Group 3 contains six acidic analytes (ESI - ); and Group 4 contains only four basic analytes. Groups 1, 2 and 3 use reversed phase chromatography on a C18 HPLC column while Group 4 uses HILIC chromatography on a bare silica column. Groups 1, 2 and 4 are analyzed in ESI + mode and Group 3 in ESI - mode. For SPE extraction, Groups 1, 2, and 3 are extracted under acidic (ph 2) conditions and Group 4 is extracted under basic (ph 10) conditions. The main aim of this work was to develop a method for EPA Method 1694 using UCT s Enviro-Clean HL DVB SPE cartridge and Selectra DA HPLC column. Another goal was to try and simplify the SPE procedure by doing a single extraction step and try to reduce the number of LC-MS/MS methods required to cover all the PCPPs. The LC-MS/MS analysis was streamlined from four methods and two HPLC columns to just two methods (ESI + and ESI - ) and a single Selectra DA HPLC column. An attempt was made to cover all the analytes in a single method using fast polarity switching, however it was found that the sensitivity of the Group 3 (ESI - ) compounds was not good when using acidic mobile phase conditions and they required a higher ph mobile phase to be efficiently ionized. However, reducing the number of LC-MS/MS methods from four to two and using a single HPLC column is significantly faster and easier than the original EPA 1694 method. Furthermore, two MRM transitions (a quantifier and a qualifier ion) were used whenever possible rather than a single MRM transition used in the EPA 1694 method The SPE procedure was optimized with the aim of obtaining acceptable recoveries for all the PCPPs using a single extraction step rather than the multiple extraction procedures outlined in the original EPA method. No sample ph adjustment of the sample was performed, which was the best compromise for the wide range of PPCPs included in the method, including for the ph sensitive β-lactam (susceptible to hydrolysis), macrolide and tetracycline antibiotics. In general, the recovery and RSD values obtained were within EPA Method 1694 requirements for the vast majority of the PPCPs included in this study. Attempts were made to try to improve the recovery of problematic analytes but this ultimately had a negative effect on some of the other PCPPs. Unfortunately, EPA Method 1694 includes a wide range of compounds that are very difficult to recover at 100% due to the wide physicochemical properties (polarity) that they possess and the stability issues experienced by some of the analytes. A comparison against the acidic and alkaline conditions outlined in EPA Method 1694 found that the recoveries of the PCPPs did not improve overall and that no ph adjustment of the water samples is the most suitable approach for a single SPE extraction procedure. The use of isotopically labeled internal standards is essential for obtaining good recoveries in EPA Method The inclusion of additional internal standards, if available, can further improve the accuracy and precision for this method, especially for difficult compounds. Furthermore, solvent standards were used for recovery calculations but the use of matrix-matched standards could further help to improve the results.
9 Conclusion: This application note outlines a simplified SPE method for the determination of a wide range of PPCPs in environmental water samples according to EPA Method Water samples are extracted using a single extraction procedure using UCT s Enviro-Clean HL DVB polymeric SPE cartridge without any sample ph adjustment. LC-MS/MS analysis was conducted with a single HPLC column (Selectra DA ) using only two methods instead of the two HPLC columns and four methods outlined in the original EPA 1694 method. The recovery and RSD values obtained were found to be within the EPA Method 1694 requirements for the vast majority of the 64 PPCPs included in this study. Overall, the streamlined screening method outlined in this application note significantly speeds up the analysis of a PPCPs in water compared to the original EPA 1694 method. The method can also be used as a starting point for a custom PPCP method that is tailored to specific matrices or compounds. In this case, further optimization of the method can be carried out to optimize results. References: [1] EPA Method 1694: Pharmaceuticals and Personal Care Products in Water, Soil, Sediment, and Biosolids by HPLC/MS/MS, December 2007, EPA-821-R UCT, LLC 2731 Bartram Road Bristol, PA methods@unitedchem.com UCT, LLC 2014 All rights reserved
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